The incidence of wound infections, especially associated with Staphylococcus aureus and Staphylococcus epidermidis, is a major concern for healthcare providers. S. aureus is one of the more common pathogens found in chronic wounds, and S. epidermidis is the most common pathogen associated with device-related infections. Further, S. pseudintermedius is a common pathogen associated with recurrent veterinary infections. S. aureus, S. epidermidis, and S. pseudintermedius are known to readily form biofilms, which are surface-adherent bacterial communities that render the bacteria resistant to ordinary antibiotics or host immune responses, and greatly increase healthcare treatment costs.
A leading factor in the pathogenesis of chronic wounds is bacterial infection. Bacteria colonizing wounds can evoke a persistent inflammatory response which is deleterious to the healing process. Cells like neutrophils and macrophages upregulate pro-inflammatory cytokines like IL-1 and TNF-α, which in turn lead to elevated levels of matrix metalloproteinases (MMPs), decreased growth factor expression, and ultimate aberration of the healing process. One of the most common species of bacteria cultured from chronic wounds is Staphylococcus aureus, which along with certain other bacterial species, has the ability to encase itself in an extracellular polysaccharide matrix (EPS) called a glycocalyx. Once a population adopts this sessile phenotype, it is substantially more resistant to host defense mechanisms as well as exogenous antimicrobials. Overcoming the protective characteristics of these “biofilms” has proven very difficult and novel effective methods of prevention and treatment are therefore very desirable.
Previous work by the present investigators indicated that zinc is required for the initial formation of staphylococcal biofilms (see, e.g. Conrady D et al. “A zinc-dependent adhesion module is responsible for intercellular adhesion in staphylococcal biofilms” PNAS 2008, 105(49):1945661, and U.S. application Ser. No. 12/994,921, the entire disclosures of which are incorporated herein by this reference.) It was shown that diethylenetriamine pentaacetic acid (DTPA), an exemplary zinc chelator, could inhibit biofilm formation by S. aureus and S. epidermidis. A biophysical characterization of a G5 domain-containing B-repeat region from Aap was disclosed, revealing that it is a zinc (Zn2+)-dependent adhesion module (“zinc adhesion module”) responsible for intercellular interaction in staphylococcal biofilms. This zinc adhesion module has been identified in a variety of bacteria, including gram positive bacteria generally, and provides a specific target for zinc chelation and biofilm inhibition in biofilms comprised of bacteria having a G5 domain. Zinc chelation was shown to inhibit formation of both S. epidermidis and methicillin-resistant S. aureus biofilms and supplementation with additional zinc in the physiological range was shown to reverse the effect. Observation of the reversible effect provides a means for identifying bacteria possessing a zinc adhesion module, which can form an adhesive intercellular contact referred to as a “zinc zipper,” and underscores the criticality of zinc in intercellular adhesion, providing a specific target for chelation and biofilm inhibition. Furthermore, it was found that addition of a soluble Aap fragment containing a single intact zinc adhesion module inhibits biofilm formation in a dose-dependent manner, indicating that the G5 domain-containing B-repeat region is indeed a required element for intercellular adhesion in staphylococcal biofilms.
Compositions comprising both an antibiotic and a metal ion chelator are known in the art. U.S. application Ser. No. 13/095,262 to Raad et al., along with a portfolio of related art out of the same laboratory, discloses compositions comprising particular antibiotics with EDTA, a well-known iron/calcium chelator. Raad restricts examples and working embodiments to compositions comprising EDTA, and teaches the necessity of alcohol in the compositions; expressly teaching that combinations of antibiotic and EDTA without alcohol require unacceptably long exposure times for desired efficacy. Raad fails to expressly disclose compositions comprising zinc chelators and fails to appreciate mechanistic underpinnings to synergies which may be achieved specifically in combinations with zinc chelators.
The use of zinc chelators in combination with antibiotic compositions is also known. U.S. patent application Ser. No. 12/391,357 to Zhang et al. discloses compositions of aminoglycoside antibiotics and ion chelators generally; however Zhang teaches addition of the ion chelator in extremely small amounts, reflecting the contemplated mechanistic role of the chelator according to Zhang. In particular Zhang teaches that a combination of β-lactam antibiotics with aminoglycoside antibiotics provides an anti-bacterial synergy, but that if dissolved in the same solution, either a salt precipitates out due to an acid-base reaction, or the amino group of the aminoglycoside antibiotic reacts with the β-lactam group of the β-lactam antibiotics, drastically reducing the efficacy of these types of antibiotics and posing potential dangers to patients if the antibiotics are administered as IV solutions. The addition of a chelator according to Zhang prevents the formation of these residual aggregate particles. Zhang did not appreciate or disclose any synergy with respect to antibiotic efficacy of an antibiotic upon addition of a chelator, or with respect to antibiotic efficacy of a chelator upon addition of an antibiotic, and did not disclose antibiotic compositions comprising amounts of chelator sufficient to provide this unexpected synergy.
Hence, there remains a need in the art for antimicrobial compositions effective against biofilms in hospital and industrial settings, and for compositions formulated to provide enhanced antibiotic efficacy in the treatment of wounds.